Betty Redfield

The properties of an E. coli ribosomal protein required for the function of factor G

Abstract Treatment of ribosomes with a solution containing 1 m NH 4 Cl and 40% ethanol removes several proteins including a factor required for factor G dependent reactions. A protein has been purified from the NH 4 Cl-ethanol extract which restores the activity of the treated ribosomes. The active factor is derived from the 50S ribosomal subunit and has a molecular weight of about 13,000.

Studies on the ribosomal sites involved in factors Tu and G-dependent reactions

Abstract Recent studies have supported the view that a common site on the ribosome may be involved in both Tu- and G-dependent reactions. The present studies have examined this problem in more detail. Thiostrepton, a known inhibitor of G-dependent reactions, inhibits the interaction of an aminoacyl-tRNA-Tu-GTP complex with 70S ribosomes. Both aminoacyl-tRNA binding and the associated GTP hydrolysis are inhibited, but nonenzymatic binding of aminoacyl-tRNA is not affected under the conditions used. There is little effect of the antibiotic on aminoacyl-tRNA binding from the ternary complex to the 30S particle, and the results show that thiostrepton inhibits binding by interacting with the 50S ribosomal subunit. Thiostrepton also inhibits, in a comparable fashion, both Tu- and G-dependent GTP hydrolysis, and these hydrolytic reactions, which are a common feature of both Tu and G activity, have been compared. The ribosomal GTP hydrolysis site shows similar although not identical heat lability in Tu- and G-dependent GTP hydrolysis. In addition, 50S ribosomal subunits which have been treated with ethanol in the presence of 1 m NH 4 Cl have much lower activity in Tu and G reactions. A protein fraction, purified from the ethanol-NH 4 Cl extract, restores the activity of the treated 50S subunit for various partial reactions catalyzed by the two transfer factors.

Further studies on the interactions of elongation factor 1 from animal tissues.

Abstract Partially purified elongation factor 1 preparations from calf brain, sheep brain, calf liver, and rabbit reticulocytes have been compared in their ability to interact with GTP and Phe-tRNA. A nitrocellulose filter assay has been used to study these interactions, and with all the EF1 preparations studied, evidence has been obtained for the formation of a Phe-tRNA·-EF1·-GTP complex. The ternary complex reacts with calf brain ribosomes in the presence of poly(U) resulting in a rapid hydrolysis of GTP and the binding of Phe-tRNA to the ribosome. Indirect evidence indicates that EF1·GDP is a product of this reaction. In the absence of poly(U) the intact complex reacts with the ribosomes without hydrolysis of GTP. The stability of the ternary complex was different with the various EF1 preparations, but the most stable complexes were prepared with calf brain EF1. Sephadex chromatography of the ternary complex shows that it contains a low molecular-weight species of the enzyme.

Purification and properties of rabbit reticulocyte elongation factor

Abstract Elongation factor 1 has been purified about 100-fold from the lysate of rabbit reticulocytes. The native enzyme is highly asymmetric ( f f 0 = 1.53 ) and has a molecular weight of 450,000. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate shows two major bands with molecular weights of about 53,000 and 50,000. Partially purified phospholipase C and AB preparations and elastase cause dissociation of the aggregate form of the enzyme to an active form which has a molecular weight of about 50,000. The effect of these phospholipase preparations is unexplained since rabbit reticulocyte elongation factor 1 contains little or no phospholipid. A protease contamination has been considered but no evidence of protease activity has been detected in the phospholipase preparations. In aminoacyl-tRNA binding, elongation factor 1 appears to show very little, if any, turnover. However, in the presence of elongation factor 2, under conditions where polymerization occurs, elongation factor 1 functions catalytically.

The binding of aminoacyl-tRNA and poly U to a soluble factor(S) extracted from ribosomes

Abstract Soluble proteins have been obtained from Escherichia coli ribosomes which have the ability to bind N-acetyl-Phe-tRNA and poly U. In addition, an interaction of a Phe-tRNA-Tu-GTP complex with the soluble extract has been demonstrated.

Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes.

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.

Aminoacyl-tRNA-Tu-GTP interaction with ribosomes.

With the aid of phosphoenolpyruvate, pyruvate kinase, and purified Phe-tRNA, a Phe-tRNA-Tu-GTP complex has been prepared which is free of unreacted Phe-tRNA. Under the conditions used in the present study, it was possible to show that during the interaction of the ternary complex with the 70S ribosome, the rate of release of Tu-GDP from the complex was faster than the formation of a stable Phe-tRNA mesenger-ribosome complex. The bulk of the Phe-tRNA bound to the ribosome from the ternary complex is on the puromycin unreactive (A) site, since factor G stimulated the formation of a puromycin product. An elevated temperature was needed for G to function in the formation of a puromycin product although at 0° it was possible to show that G reacted with the ribosome and that GTP hydrolysis occurred. Studies on the interaction of the Phe-tRNA-Tu-GTP complex with 30S ribosomal subunit required a heat activation of the 30S particle. The heat activation was not needed for the binding of poly U to the 30S particle.

Tetrazolium reduction as a measure of monoamine oxidase activity in vitro.

1 . Data has been presented showing that tetrazolium salts can be used for the histochemical localization of monoamine oxidase, although there are limitations to its application to in vitro studies. These limitations have been discussed. 2. It appears that an indole carbonyl compound is necessary for 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction. A diaphorasetype system may be required to transfer the electrons from the indole carbonyl intermediate to INT.

Further studies on the role of factors Ts and Tu in protein synthesis.

Abstract A factor Ts stimulation for amino acid polymerization is observed only in the presence of low levels of factor Tu. The Ts dependency that is obtained under these conditions can be replaced to the extent of 60–80% by phosphoenolpyruvate and pyruvate kinase. These results support the view that the primary action of Ts in polymerization is to catalyze the exchange reaction shown below which results in the formation of Tu-GTP, the species of Tu which reacts with aminoacyl-tRNA. Tu-GDP + GTP /af Tu-GTP + GDP The lack of Ts dependency in the presence of high levels of Tu-GDP may be explained by a low rate of nucleotide exchange that occurs in the absence of Ts. Both factors Ts and Tu function catalytically in the polymerization reaction.

Studies on the reaction of the aminoacyl-tRNA-Tu-GTP complex with ribosomal subunits

Abstract A Phe-tRNA-Tu-GTP complex binds to the 30S ribosomal subunit in the presence of poly U without appreciable hydrolysis of GTP. Under the conditions used, little binding is seen with the 50S ribosomal subunit. However, in the presence of poly U and both subunits, the rate of binding of aminoacyl-tRNA is markedly increased and hydrolysis of the GTP in the complex occurs.